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, 
, ¶, **, **
*State Key Lab for Medical Genomics, Shanghai Institute of
Hematology, Ruijin Hospital Affiliated to Shanghai Second Medical University,
Shanghai 200025, China; Health Science Center, Shanghai Second Medical University and the
Shanghai Institute of Biological Science, Chinese Academy of Sciences, Shanghai
200025, China; Department of Pediatric Oncology, Dana-Farber Cancer Institute,
Boston, MA 02115; and ¶Division of Hematology and Oncology and
||Howard Hughes Medical Institute, Children's Hospital and Harvard
Medical School, Boston, MA 02115
Contributed by Zhu Chen, October 1, 2004
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Abstract |
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The zebrafish kidney marrow is considered to be the organ of definitive hematopoiesis, analogous to the mammalian bone marrow. We have sequenced 26,143 ESTs and isolated 304 cDNAs with putative full-length ORF from a zebrafish kidney marrow cDNA library. The ESTs formed 7,742 assemblies, representing both previously identified zebrafish ESTs (56%) and recently discovered zebrafish ESTs (44%). About 30% of these EST assemblies have orthologues in humans, including 1,282 disease-associated genes in the Online Mendelian Inheritance in Man (OMIM) database. Comparison of the effective and regulatory molecules related to erythroid functions across species suggests a good conservation from zebrafish to human. Interestingly, both embryonic and adult zebrafish globin genes showed higher homology to the human embryonic globin genes than to the human fetal/adult ones, consistent with evo-devo correlation hypothesis. In addition, conservation of a whole set of transcription factors involved in globin gene switch suggests the regulatory network for such remodeling mechanism existed before the divergence of the teleost and the ancestor of mammals. We also carried out whole-mount mRNA in situ hybridization assays for 493 cDNAs and identified 80 genes (16%) with tissue-specific expression during the first five days of zebrafish development. Twenty-six of these genes were specifically expressed in hematopoietic or vascular tissues, including three previously unidentified zebrafish genes: coro1a, nephrosin, and dab2. Our results indicate that conserved genetic programs regulate vertebrate hematopoiesis and vasculogenesis, and support the role of the zebrafish as an important animal model for studying both normal development and the molecular pathogenesis of human blood diseases.
Cataloging ESTs and isolation of full-length cDNAs are essential means for gene discovery, as well as for the understanding of tissue/organ functions and the identification of tissue-specific markers. The comparative studies of transcriptomes among different species also shed light on the evolution of the molecular mechanism in physiology. Meanwhile, whole-mount mRNA in situ hybridization (WISH) in zebrafish has been used as a common assay to explore gene function and to find useful probes in mutagenesis screening (5, 6). To date, >26 complementation groups of mutations have been identified in zebrafish that affect hematopoietic development (1). The characterizations of these mutant lines have shown that most of them affect critical genes within the erythroid pathway (1, 7). However, few mutants that specifically affect granulopoiesis have been identified because of the paucity of zebrafish granulocyte-specific markers (2).
In this study, an analysis of zebrafish kidney marrow EST and full-length ORF-containing cDNA clones provided some insights into the evolutionary features of genetic regulation of hematopoiesis in vertebrates. The WISH assay uncovered the spatial and temporal expression patterns of 80 genes exhibiting tissue-specific expression during embryogenesis. In addition, we have focused on the developmental expression pattern of three recently discovered zebrafish genes in granulocytic and vascular tissues, coro1a, nephrosin, and dab2, whose roles in mammalian hematopoiesis and vasculature development are not yet well defined.
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Materials and Methods |
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ZAP expression vector,
PBK-CMV (Stratagene), at the XhoI and EcoRI sites. The
ligated products were packaged by using Gigapack Gold packaging
extracts. Bacteria growth and plasmid extractions were performed in a
96-well format (Qiagen, Valencia, CA). Sequencing reactions were
performed with T3 (5' end) and T7 (3' end) primers on 9600 Thermal
Reactors (PerkinElmer). The reaction products were analyzed by using
an ABI 377 or ABI 3700 DNA Sequencer (Applied Biosystems) as
described (9).
Bioinformatic Analysis and Data Management. Quality assessment and base trimming of ESTs were performed by using PHRED2.1. The quality of the sequences was judged "good" if they contained <3% ambiguous bases and were >100 bp. EST sequences were clustered by CAT3.5 software (Pangea, Oakland, CA) to obtain EST assemblies, which were sequentially searched against the zebrafish UniGene and dbEST databases (http://www.ncbi.nlm.nih.gov/). The EST assembly was considered identical to known entries (ESTs or genes) if they shared at least 95% identity over 100 bp of DNA sequences. The prediction of zebrafish-human orthologues relied on the "reciprocal best hit" search methods as described (10, 11). The criteria for a cDNA to be accepted as having an entire ORF have been described (12). The programs SIGNALP and TMHMM were used to predict the signal peptide and the transmembrane domains of full-length cDNAs.
Fish Maintenance and Embryonic Staging. AB zebrafish were maintained and staged as described (13). Embryos raised to time points beyond 24 h postfertilization (hpf) were treated with 0.003% phenylthiourea to prevent melanization. Embryos were removed from their outer chorions with 0.001% pronase and fixed overnight at 18, 24, 72, and 120 hpf in 4% paraformaldehyde at 4°C. Fixed embryos were washed in PBST (PBS supplemented with 0.1% Tween 20) and dehydrated in graded PBST/methanol solutions (3:1, 1:1, 1:3) for 10 min each and stored in absolute methanol at -20°C.
WISH and Histology. Fixed embryos (stored at -20°C in methanol) were rehydrated in graded PBST/methanol solutions (1:3, 1:1, 3:1) for 10 min each, followed by PBST rinse twice for 10 min each at room temperature (RT). Embryos at 72 and 120 hpf were further permeabilized with proteinase K solution (100 µg/ml) at RT for 20-30 min, rinsed in PBST twice, and refixed in 4% paraformaldehyde at RT for 30 min. Embryos at 18 and 24 hpf were not treated with proteinase K. Ten to 15 embryos from each time points (18, 24, 72, and 120 hpf) were combined and hybridized with digoxigenin-labeled antisense RNA probes at 68°C. Synthesis of RNA probes and hybridizations were performed as described (2). Double in situ hybridization assays were performed by using digoxigenin and fluorescein-labeled probes, and developed with the chromagenic substrates 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium and Fast Red. After in situ hybridization, embryos were rehydrated and embedded in 3% sucrose overnight at 4°C before cryostat sectioning. Sections were counterstained with eosin and photographed.
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Results |
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Sequence-based "reciprocal best hit" analysis provides a reliable method to identify zebrafish orthologues of human genes (10). Of 7,742 unique transcripts, 2,225 (28.7%) were considered to be zebrafish orthologues of human genes, of which 272 were previously entered into zebrafish public databases and 1,953 were not. The 2,225 orthologues were grouped into seven categories based on the general cellular function, including cell signaling/communication (19.4%), gene/protein expression (18.7%), metabolism (14.6%), cell structure/motility (5.5%), cell division (4.7%), and cell organism/defense (4.2%), with the remaining 33% lacking enough structural and functional information for classification. Of note, 1,282 of these orthologues corresponded to human disease genes as registered in the Online Mendelian Inheritance in Man (OMIM) database. In addition, 304 previously undescribed cDNA clones with complete ORF were characterized based on our EST source, with a range of ORF length of 68-1,235 aa (median, 373 ± 10.6 aa). These genes might be involved in critical biological process according to their homology to known genes with established functions, such as secretory proteins, transmembrane proteins, signal transduction molecules, and enzymes, etc. (see Table 2, which is published as supporting information on the PNAS web site, and www.chgc.org.cn/danio).
Hematopoietic and Vascular Genes. Ninety genes (Table 3, which is published as supporting information on the PNAS web site), whose mammalian homologues have been shown to be either associated functionally with hematopoiesis/angiogenesis or hematopoietic disorder, were divided into three subgroups: (i) genes regulating the processes of hematopoiesis or angiogenesis (regulators); (ii) genes related to mature blood cell function (effectors), and (iii) genes involved in human hematopoietic disorders. These genes include cytokines, receptors, signal transduction molecules, and transcription factors that regulate the processes of blood or blood vessel development in different stages. It is worth noting that some genes involved in human hematopoietic diseases were uncovered in the present work. For example, the human CRKL gene has been implicated as a substrate for the BCR-ABL tyrosine kinase in chronic myelogenous leukemia (14), and six leukemia-associated genes, MSF, SEPT6, FNBP1, GAS7, ARHGEF12 and ELL, have been identified as components of MLL fusion proteins in leukemia with 11q23 chromosomal translocation (15-20).
Many mammalian genes have been shown to play a role in the development
and maturation of hematopoietic and vascular progenitors (Fig. 1)
(7,
21). In
this study, 21 orthologues of these genes were identified: two
(smad5 and sdf1) are involved in the induction of blood
from ventral mesoderm (7, 22); nine
(c-myb, scl, fli1, flk1, pu.1, cebp
, cebp
,
ikaros, and klf1) are required for the maintenance and
commitment of hematopoietic stem/progenitor cells; and six
[hemoglobin (hbaa1, ba1, ba2), alas2, myeloperoxidase
(mpo), and phox47] are expressed specifically in the
erythroid or granulocytic lineages. In addition, a number of genes
encoding homologues of human cytokines, jak1, stat3, raf, erk1,
pim1, and ikb (data not shown), which
act as signaling transducers required for granulopoeisis, are
present.
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- and -like families (Fig.
2A), and, in phylogenetic analysis, they formed distinct
clusters with those of other species. Nevertheless, within each
family, although the same set of the embryonic/fetal/adult globin
genes from mammals constituted monophyletic branch, the zebrafish
embryonic and adult globin subtypes found no such kind of close
relationship with mammals and could only form an independent
branch. These data suggest that the ancestors of - or -like globins
should exist before the divergence between the teleost and the
ancestor of mammals 
450 million years ago,
whereas the differentiation of subtype globin genes with regard to
developmental stages might occur after this divergence. A pair-wise
analysis of the similarity between zebrafish and human globins was
further examined. The result revealed that the zebrafish globins
consistently maintained highest homology to these human globin genes
that were early expressed during embryogenesis. For example, the
zebrafish -like globins (hbael, hbae3, hbaa1) were most similar to the
human 
globin, whereas zebrafish -like globins
(hbbel, hbbe2, hbbe3, bal, ba2) displayed strongest homology to
human 
globin (Table 1). It
may be postulated, therefore, that embryonic globin genes represent
the evolutionarily most conserved ones and the zebrafish globins may
be have functionally more like the human embryonic globins, a point
of view consistent with the evo-devo correlation hypothesis (23) and in
strong support of the previous speculation that the individual member
genes of each globin family have arisen through a series of
tandem duplications of ancestral - and -like globin genes during
the evolution of higher vertebrates (24, 25).
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globin, and it also inhibits the expression of globin
gene in the presence of YY1, whereas KLF1 enhances the
expression of globin gene. Our analysis showed that all these regulators
are well conserved in zebrafish (Fig. 2
C-H), indicating an early existence of regulatory
network for remodeling mechanism such as the globin switch. It also
may be interesting to note that, according to their position in the
phylogenetic trees, regulators for early switch (GATA1, YY1,
NR2F2, NFE2, and TIEG2) have higher homology through
evolution than that of KLF1, a regulator of second subtype
switch (Fig. 2
C-H). Interestingly, like the recent report for
another fish species, the four subtype of T cell receptor genes
(, , , 
) were found in zebrafish and showed close relationship with
the corresponding human genes (data not shown), suggesting that the
major hematopoiesis-related molecules appeared in a very early stage
of vertebrate evolution.
WISH Analysis of Gene Expression. Because the functions of many of the mammalian homologues of the zebrafish ESTs are poorly understood, the examination of their respective gene expression patterns during early development should help to establish their tissue-specific roles and to identify valuable reagents for dissecting hematopoietic regulatory pathways. A total of 493 unique EST assemblies were selected based on their potential relevance to human hematopoiesis and diseases for WISH analysis. Embryos at 18, 24, 72, and 120 hpf were used to detect the dynamic changes in gene expression that occur during embryonic organogenesis. Fig. 3Aa illustrates the three major spatial and temporal expression patterns that have been observed: (i) no signal at any of the four time points (148 of 493; 30%); (ii) ubiquitous expression at all four time points (265 of 493; 54%); and (iii) tissue-specific gene expression in at least one embryonic stage (80 of 493; 16%). For examples, the expression of phenylalanine hydroxylase (pah), whose deficiency causes phenylketonuria and mental retardation in human infants, was detected in the developing central nervous system (CNS) and pronephric ducts (pnd) at 18 (Fig. 3A b-d) and 24 (Fig. 3A e and f) hpf. A number of genes were found exclusively expressed in the developing embryonic kidney, such as zebrafish renin in the putative juxtaglomerular cells (Fig. 3A g-j) or a recently discovered EST (RK011A3G03) in the pnd (Fig. 3A k-n). Other genes exhibited specific expression patterns in other organs/tissues such as the central nervous system, thyroid, skin, liver, ears, and neuromasts (see Tables 2 and 3 and www.chgc.org.cn/danio).
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. However, the
significance of several clones expressed in blood or vasculature,
such as coro1a (GenBank accession no. AY330221
[GenBank]
), nephrosin (GenBank accession no. AY330222
[GenBank]
), and dab2 (GenBank accession no. AY330223
[GenBank]
), remained to be established. We have thus
examined the expression patterns of these genes in more detail.
Recently Discovered Zebrafish Myeloid-Restricted Genes coro1a and nephrosin. The zebrafish orthologue of human CORO1A gene, coro1a, was expressed in cells that migrated over the surface of the anterior head and yolk at 18 hpf (Fig. 3B a and b) and throughout the embryo by 24 hpf (Fig. 3Bc). Double WISH assays with both mpo, a specific marker for both mammalian and zebrafish neutrophils, and coro1a were performed on 24-hpf embryos. All mpo-positive cells were also coro1a-positive, whereas some cells expressed coro1a alone (Fig. 3B d and e).
The zebrafish nephrosin-positive cells were first found scattered
over the surface of anterior yolk in 10% of 24-hpf embryos.
By 30 hpf, all embryos contained cells expressing nephrosin
in both anterior yolk and posterior ICM (data not shown). By 72
and 120 hpf, cells expressing zebrafish nephrosin were distributed
throughout the embryo, but concentrated over the anterior yolk,
in the posterior ICM and in the posterior blood island (Fig.
3B f and g). Double WISH assays indicated that most
nephrosin-positive cells also expressed the granulocytic
marker mpo (Fig.
3Bh), whereas a smaller number of these cells coexpressed
the monocytic lysozyme c at 72 hpf (Fig.
3Bi). The nephrosin-positive cells did not
coexpress c-myb transcripts in 72-hpf embryos (data not
shown), suggesting that nephrosin was not expressed by myeloid
progenitors. Compared to some granulocyte-specific genes such
as mpo expressed as early as 18 hpf, zebrafish nephrosin
could be a relatively late stage marker of granulocytic
differentiation.
Recently Discovered Zebrafish Vascular-Specific Gene dab2. The expression of zebrafish dab2 was restricted to cells of the pnd, ICM, dorsal aorta (da), and caudal vein (cv) of 18- and 24-hpf embryos (Fig. 3C a and b). By 3 days after fertilization, expression of dab2 was detected mainly in cv (Fig. 3Cc) and pnd (Fig. 3Cd) as shown in cross section (Fig. 3Ce) at the level indicated in Fig. 3Cd. Double WISH studies on 24-hpf embryos with fli1 confirmed that dab2 was coexpressed with zebrafish fli1 in da and cv cells forming in the trunk, but not in the most posterior cells of the developing posterior blood island (Fig. 3C f and g, arrows). In contrast, assays for the coexpression of dab2 with the stem cell gene scl showed that these two genes were not coexpressed in cells of the ICM, or in the developing vasculture (Fig. 3C h and i, arrows).
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Discussion |
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360,000 ESTs, whereas the
zebrafish Unigene database had 17,000 unique gene entries.
In our work, 7,742 EST assemblies of unique sequences and 304
full-length cDNAs were identified from a cDNA library of the
zebrafish kidney marrow. Because 44% of these EST assemblies were
previously undescribed ones, this study expands both the current
zebrafish UniGene and EST databases. Furthermore, we identified
2,225 zebrafish orthologues of human genes, providing useful
means to perform further functional analysis of these genes or
to compare certain tissue-restricted pathways.
The gene expression profile of a given organ/tissue should reflect
its physiological functions. As expected, many orthologues of
mammalian genes required for blood and vasculature development
are expressed in the zebrafish kidney marrow cDNA library. In
general, these orthologues show a very high level of homology
and are thought to be involved at different stages of differentiation
along with distinct hematopoietic lineages. These findings have
provided important molecular evidence for the conservation of
genetic programs that regulate vertebrate hematopoiesis and
confirmed that the adult kidney marrow is the major site of
definitive hematopoiesis in the zebrafish. Of particular interest
is the characterization of some evolutionary features of the
embryonic/fetal/adult globin genes and related transcription
factors between zebrafish and mammals. It seems that the ancestors
for both - and -globin families have existed before the divergence of the
common ancestor of teleost and mammals, whereas distinct globin gene
subtypes in each family might have evolved after this divergence,
because no corresponding phylogenetic relationship could be seen
among embryonic and adult globins between zebrafish and mammals. On
the other hand, the fact that zebrafish globin genes in each family
exhibited higher homology to embryonic, but not adult, counterparts
in human suggests that these embryonic globin genes should be the
evolutionarily most conserved ones. Moreover, the conservation of the
transcription factors for globin gene switch across species from
zebrafish to man is in support of the view that the regulatory
mechanism underlying globin expression remodeling should exist at
very early stage of vertebrate evolution.
Previous studies showed that WISH could provide important data to
aid understanding gene function. As the first step in an effort to
carry out systematic WISH analysis for zebrafish gene expression, we
conducted an investigation of genes expressed in zebrafish kidney
marrow. Detailed description was made in this study for three
zebrafish homologues, coro1a, nephrosin, and dab2, that
exhibited specific expression in hematopoietic/vascular tissues, but
whose functions in mammals are poorly understood. Although there is
currently no functional data on human CORO1A, mouse Coro1A has been
shown to contain a WD (trp/asp) repeat and a leucine zipper motif (26) and to
reside on phagosomal membranes in cells infected by mycobacteria,
thus preventing endocytic transport of these bacteria to lysosomes
and promoting their intracellular survival in cultured macrophages
(27).
This observation is consistent with our results that coro1a is
expressed in myeloid cells coexpressing mpo and indicates that
coro1a is a marker for mature granulocyte and monocytes. The
nephrosin gene has not yet been identified in mammals, but has
been found in carp (Cyprinus carpio). Carp nephrosin is a
secreted zinc-coordinating endopeptidase of the astacin family (28). In
zebrafish, nephrosin is expressed specifically in
granulocytes. One could speculate that this protease may function by
its secretion into the extracellular matrix, potentially playing a
role in either the migration of myeloid cells or their ability to
invade other tissues. In support of this hypothesis, carp
nephrosin has been reported to be able to degrade both
mammalian fibronectin and gelatin (28). The
colocalization of zebrafish dab2 with fli1 expression in
developing zebrafish vasculature is an intriguing finding. Human
DAB2 is a candidate tumor suppressor gene in epithelial
ovarian cancer (29) and links
transforming growth factor receptors to the
Smad pathway in a fibrosarcoma cell line (30). In
zebrafish, dab2 expression is observed in presumptive
endothelial cells of the forming vasculature. Although colocalization
of dab2 with fli1 in the dorsal aorta and axial vein of
trunk in 18- to 24-hpf embryos was evident, several differences were
also observed. The fli1 gene is expressed in ventral mesoderm
by 12 hpf, whereas dab2 is not expressed until later (data
not shown). In addition, the fli1 gene is expressed in the
head of 24-hpf embryos and the developing posterior blood
island, whereas dab2 expression appears to be restricted to
the developing vasculature of the trunk. These results suggest that
dab2 is a bona fide trunk vascular marker during
embryogenesis. Taken as a whole, our results demonstrated once again
the value of WISH analysis of zebrafish genes as a unique approach in
comparative functional genomics.
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Acknowledgements |
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Footnotes |
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Data deposition: The sequences reported in this paper have been deposited in the GenBank database (accession nos. CD580595 [GenBank] -CD606737, AY391404 [GenBank] -AY391473, AY394926 [GenBank] -AY394976, AY398305 [GenBank] -AY398433, AY422992 [GenBank] -AY423041, and AY330221 [GenBank] -AY330224).
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H.-D.S., X.-J.S., M.D., G.-W.Z., Y.Z., and X.-Y.W.
contributed equally to this work. 
** To whom correspondence may be addressed. E-mail: zchen{at}stn.sh.cn or thomas_look{at}dfci.harvard.edu .
© 2004 by The National Academy of Sciences of the USA
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